Every cell division requires the faithful duplication of genetic material from mother cell to daughter cell. Defects in the proper execution of the DNA replication program can directly result in the genome instability that is a hallmark of nearly all cancer cells. Proper genome duplication requires the delicate balance of the initiation and elongation phases of DNA replication. In fact, it has recently been demonstrated that the breakdown of replication forks within large genomic regions incapable of initiating DNA replication is responsible for the generation of chromosomal fragile sites, or sites in the genome susceptible to breakage. Our recent work in Drosophila has identified regions of the genome that are selectively repressed for DNA replication in a tissue-specific manner, providing a framework to understand the causes of tissue-specific genome instability. Furthermore, this repression is due to inhibition of replication fork progression through regions of the genome that are unable to initiate DNA replication. A single chromatin protein has been shown to mediate this tissue-specific repression, emphasizing the effect of chromatin structure on replication fork progression and stability. Therefore, we have now established a model system to understand how developmental changes in the DNA replication program ultimately affect genome stability and lead to chromosome fragile sites. Thus, the major goals of this proposal are to understand how chromatin structure influences replication fork progression and stability, and to identify components of active replication forks that mediate their progression through diverse chromatin structures.